RESUMO
To survive extreme desiccation, seeds enter a period of quiescence that can last millennia. Seed quiescence involves the accumulation of protective storage proteins and lipids through unknown adjustments in protein homeostasis (proteostasis). Here, we show that mutation of all six type-II metacaspase (MCA-II) proteases in Arabidopsis thaliana disturbs proteostasis in seeds. MCA-II mutant seeds fail to restrict the AAA ATPase CELL DIVISION CYCLE 48 (CDC48) at the endoplasmic reticulum to discard misfolded proteins, compromising seed storability. Endoplasmic reticulum (ER) localization of CDC48 relies on the MCA-IIs-dependent cleavage of PUX10 (ubiquitination regulatory X domain-containing 10), the adaptor protein responsible for titrating CDC48 to lipid droplets. PUX10 cleavage enables the shuttling of CDC48 between lipid droplets and the ER, providing an important regulatory mechanism sustaining spatiotemporal proteolysis, lipid droplet dynamics, and protein homeostasis. In turn, the removal of the PUX10 adaptor in MCA-II mutant seeds partially restores proteostasis, CDC48 localization, and lipid droplet dynamics prolonging seed lifespan. Taken together, we uncover a proteolytic module conferring seed longevity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Retículo Endoplasmático , Gotículas Lipídicas , Mutação , Sementes , Proteína com Valosina , Arabidopsis/genética , Arabidopsis/metabolismo , Sementes/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Retículo Endoplasmático/metabolismo , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Gotículas Lipídicas/metabolismo , Proteostase , Proteólise , Regulação da Expressão Gênica de Plantas , Longevidade/fisiologia , Longevidade/genéticaRESUMO
This study uncovers a regulatory interplay between WRINKLED1 (WRI1), a master transcription factor for glycolysis and lipid biosynthesis, and Translocator Protein (TSPO) expression in Arabidopsis thaliana seeds. We identified potential WRI1-responsive elements upstream of AtTSPO through bioinformatics, suggesting WRI1's involvement in regulating TSPO expression. Our analyses showed a significant reduction in AtTSPO levels in wri1 mutant seeds compared to wild type, establishing a functional link between WRI1 and TSPO. This connection extends to the coordination of seed development and lipid metabolism, with both WRI1 and AtTSPO levels decreasing post-imbibition, indicating their roles in seed physiology. Further investigations into TSPO's impact on fatty acid synthesis revealed that TSPO misexpression alters WRI1's post-translational modifications and significantly enhances seed oil content. Additionally, we noted a decrease in key reserve proteins, including 12 S globulin and oleosin 1, in seeds with TSPO misexpression, suggesting a novel energy storage strategy in these lines. Our findings reveal a sophisticated network involving WRI1 and AtTSPO, highlighting their crucial contributions to seed development, lipid metabolism, and the modulation of energy storage mechanisms in Arabidopsis.
RESUMO
Since decades plant lipid droplets (LDs) are described as storage organelles accumulated in seeds to provide energy for seedling growth after germination. Indeed, LDs are the site of accumulation for neutral lipids, predominantly triacylglycerols (TAGs), one of the most energy-dense molecules, and sterol esters. Such organelles are present in the whole plant kingdom, from microalgae to perennial trees, and can probably be found in all plant tissues. Several studies over the past decade have revealed that LDs are not merely simple energy storage compartments, but also dynamic structures involved in diverse cellular processes like membrane remodeling, regulation of energy homeostasis and stress responses. In this review, we aim to highlight the functions of LDs in plant development and response to environmental changes. In particular, we tackle the fate and roles of LDs during the plant post-stress recovery phase.
RESUMO
Autophagy is a universal mechanism that facilitates the degradation of unwanted cytoplasmic components in eukaryotic cells. In this review, we highlight recent developments in the investigation of the role of autophagy in lipid homeostasis in plants by comparison with algae, yeast, and animals. We consider the storage compartments that form the sources of lipids in plants, and the roles that autophagy plays in the synthesis of triacylglycerols and in the formation and maintenance of lipid droplets. We also consider the relationship between lipids and the biogenesis of autophagosomes, and the role of autophagy in the degradation of lipids in plants.
Assuntos
Autofagia , Gotículas Lipídicas , Animais , Autofagossomos , Lipídeos , PlantasRESUMO
During the life cycle of plants, seedlings are considered vulnerable because they are at the interface between the highly stress tolerant seed embryos and the established plant, and must develop rapidly, often in a challenging environment, with limited access to nutrients and light. Using a simple experimental system, whereby the seedling stage of Arabidopsis is considerably prolonged by nutrient starvation, we analysed the physiology and metabolism of seedlings maintained in such conditions up to 4 weeks. Although development was arrested at the cotyledon stage, there was no sign of senescence and seedlings remained viable for weeks, yielding normal plants after transplantation. Photosynthetic activity compensated for respiratory carbon losses, and energy dissipation by photorespiration and alternative oxidase appeared important. Photosynthates were essentially stored as organic acids, while the pool of free amino acids remained stable. Seedlings lost the capacity to store lipids in cytosolic lipid droplets, but developed large plastoglobuli. Arabidopsis seedlings arrested in their development because of mineral starvation displayed therefore a remarkable resilience, using their metabolic and physiological plasticity to maintain a steady state for weeks, allowing resumption of development when favourable conditions ensue.
Assuntos
Arabidopsis/fisiologia , Estresse Fisiológico , Arabidopsis/metabolismo , Metabolismo dos Lipídeos , Minerais/metabolismo , Modelos Biológicos , Plântula/metabolismo , Plântula/fisiologiaRESUMO
Postgerminative mobilization of neutral lipids stored in seed lipid droplets (LDs) is preceded by the degradation of oleosins, the major structural LD proteins that stabilize LDs in dry seeds. We previously showed that Arabidopsis thaliana oleosins are marked for degradation by ubiquitination and are extracted from LDs before proteolysis. However, the mechanisms underlying the dislocation of these LD-anchored proteins from the LD monolayer are yet unknown. Here, we report that PUX10, a member of the plant UBX-domain containing (PUX) protein family, is an integral LD protein that associates with a subpopulation of LDs during seed germination. In pux10 mutant seedlings, PUX10 deficiency impaired the degradation of ubiquitinated oleosins and prevented the extraction of ubiquitinated oleosins from LDs. We also showed that PUX10 interacts with ubiquitin and CDC48A, the AAA ATPase Cell Division Cycle 48, through its UBA and UBX domains, respectively. Collectively, these results strongly suggest that PUX10 is an adaptor recruiting CDC48A to ubiquitinated oleosins, thus facilitating the dislocation of oleosins from LDs by the segregase activity of CDC48A. We propose that PUX10 and CDC48A are core components of a LD-associated degradation machinery, which we named the LD-associated degradation system. Importantly, PUX10 is also the first determinant of a LD subpopulation described in plants, suggesting functional differentiation of LDs in Arabidopsis seedlings.
Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Gotículas Lipídicas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Mutação , Sementes/metabolismo , Ubiquitina/metabolismoRESUMO
Cytosolic lipid droplets are dynamic lipid-storage organelles that play a crucial role as reservoirs of metabolic energy and membrane precursors. These organelles are present in virtually all cell types, from unicellular to pluricellular organisms. Despite similar structural organization, lipid droplets are heterogeneous in morphology, distribution and composition. The protein repertoire associated to lipid droplet controls the organelle dynamics. Distinct structural lipid droplet proteins are associated to specific lipolytic pathways. The role of these structural lipid droplet-associated proteins in the control of lipid droplet degradation and lipid store mobilization is discussed. The control of the strictly-regulated lipolysis in lipid-storing tissues is compared between mammals and plants. Differences in the cellular regulation of lipolysis between lipid-storing tissues and other cell types are also discussed.
Assuntos
Membrana Celular/metabolismo , Gotículas Lipídicas/metabolismo , Lipólise/fisiologia , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Animais , HumanosRESUMO
In oleaginous seeds, lipids--stored in organelles called oil bodies (OBs)--are degraded post-germinatively to provide carbon and energy for seedling growth. To date, little is known about how OB coat proteins, known as oleosins, control OB dynamics during seed germination. Here, we demonstrated that the sequential proteolysis of the five Arabidopsis thaliana oleosins OLE1-OLE5 begins just prior to lipid degradation. Several post-translational modifications (e.g. phosphorylation and ubiquination) of oleosins were concomitant with oleosin degradation. Phosphorylation occurred only on the minor OLE5 and on an 8 kDa proteolytic fragment of OLE2. A combination of immunochemical and proteomic approaches revealed ubiquitination of the four oleosins OLE1-OLE4 at the onset of OB mobilization. Ubiquitination topology was surprisingly complex. OLE1 and OLE2 were modified by three distinct and predominantly exclusive motifs: monoubiquitin, K48-linked diubiquitin (K48Ub(2)) and K63-linked diubiquitin. Ubiquitinated oleosins may be channeled towards specific degradation pathways according to ubiquitination type. One of these pathways was identified as the ubiquitin-proteasome pathway. A proteasome inhibitor (MG132) reduced oleosin degradation and induced cytosolic accumulation of K48Ub(2)-oleosin aggregates. These results indicate that K48Ub(2)-modified oleosins are selectively extracted from OB coat and degraded by the proteasome. Proteasome inhibition also reduced lipid hydrolysis, providing in vivo evidence that oleosin degradation is required for lipid mobilization.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Gotículas Lipídicas/metabolismo , Plântula/metabolismo , Ubiquitina/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Inibidores de Cisteína Proteinase/farmacologia , Germinação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Leupeptinas/farmacologia , Microscopia Confocal , Fosforilação , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteólise/efeitos dos fármacos , Proteômica/métodos , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Fatores de Tempo , UbiquitinaçãoRESUMO
Oil bodies (OBs) are seed-specific lipid storage organelles that allow the accumulation of neutral lipids that sustain plantlet development after the onset of germination. OBs are covered with specific proteins embedded in a single layer of phospholipids. Using fluorescent dyes and confocal microscopy, we monitored the dynamics of OBs in living Arabidopsis (Arabidopsis thaliana) embryos at different stages of development. Analyses were carried out with different genotypes: the wild type and three mutants affected in the accumulation of various oleosins (OLE1, OLE2, and OLE4), three major OB proteins. Image acquisition was followed by a detailed statistical analysis of OB size and distribution during seed development in the four dimensions (x, y, z, and t). Our results indicate that OB size increases sharply during seed maturation, in part by OB fusion, and then decreases until the end of the maturation process. In single, double, and triple mutant backgrounds, the size and spatial distribution of OBs are modified, affecting in turn the total lipid content, which suggests that the oleosins studied have specific functions in the dynamics of lipid accumulation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriologia , Arabidopsis/metabolismo , Corpos de Inclusão/metabolismo , Óleos de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Germinação , Imageamento Tridimensional , Fenótipo , Análise de Regressão , Coloração e RotulagemRESUMO
Oleaginous seeds store lipids in specialized structures called oil bodies (OBs). These organelles consist of a core of neutral lipids bound by proteins embedded in a phospholipid monolayer. OB proteins are well conserved in plants and have long been grouped into only two categories: structural proteins or enzymes. Recent work, however, which identified other classes of proteins associated with OBs, clearly shows that this classification is obsolete. Proteomics-mediated OB protein identification is facilitated in plants for which the genome is sequenced and annotated. However, it is not clear whether this knowledge can be dependably transposed to less well-characterized plants, including the well-established commercial sources of seed oil as well as the many others being proposed as novel sources for biodiesel, especially in Africa and Asia. Toward an update of the current data available on OB proteins this review discusses (i) the specific difficulties for proteomic studies of organelles; (ii) a 2012 census of the proteins found in seed OBs from various crops; (iii) the oleosin composition of OBs and their role in organelle stability; (iv) PTM of OB proteins as an emerging field of investigation; and finally we describe the emerging model of the OB proteome from oilseed crops.
Assuntos
Produtos Agrícolas , Óleos de Plantas , Proteínas de Plantas , Sementes , Processamento de Proteína Pós-Traducional , ProteomaRESUMO
Despite the importance of seed oil bodies (OBs) as enclosed compartments for oil storage, little is known about lipid and protein accumulation in OBs during seed formation. OBs from rapeseed (Brassica napus) consist of a triacylglycerol (TAG) core surrounded by a phospholipid monolayer embedded with integral proteins which confer high stability to OBs in the mature dry seed. In the present study, we investigated lipid and protein accumulation patterns throughout seed development (from 5 to 65 days after pollination [DAP]) both in the whole seed and in purified OBs. Deposition of the major proteins (oleosins, caleosins and steroleosins) into OBs was assessed through (i) gene expression pattern, (ii) proteomics analysis, and (iii) protein immunodetection. For the first time, a sequential deposition of integral OB proteins was established. Accumulation of oleosins and caleosins was observed starting from early stages of seed development (12-17 DAP), while steroleosins accumulated later (~25 DAP) onwards.
Assuntos
Brassica napus/metabolismo , Óleos de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Brassica napus/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Ácidos Graxos/metabolismo , Expressão Gênica , Fosfolipídeos/metabolismo , Proteínas de Plantas/genética , Proteômica , Sementes/crescimento & desenvolvimento , Fatores de Tempo , Triglicerídeos/metabolismoRESUMO
In 2000, Marquardt et al. (A. Marquardt, H. Stöhr, K. White, and B. H. F. Weber. 2000. cDNA cloning, genomic structure, and chromosomal localization of three members of the human fatty acid desaturase family. Genomics. 66: 176-183.) described the genomic structure of the fatty acid desaturase (FADS) cluster in humans. This cluster includes the FADS1 and FADS2 genes encoding, respectively, for the Delta 5- and Delta 6-desaturases involved in polyunsaturated fatty acid biosynthesis. A third gene, named FADS3, has recently been identified but no functional role has yet been attributed to the putative FADS3 protein. In this study, we investigated the FADS3 occurrence in rat tissues by using two specific polyclonal antibodies directed against the N-terminal and C-terminal ends of rat FADS3. Our results showed three potential protein isoforms of FADS3 (75 kDa, 51 kDa, and 37 kDa) present in a tissue-dependent manner. The occurrence of these FADS3 isoforms did not depend on the mRNA level determined by real-time PCR. In parallel, mouse tissues were also tested and showed the same three FADS3 isoforms but with a different tissue distribution. Finally, we reported the existence of FADS3 in human cells and tissues but different new isoforms were identified. To conclude, we showed in this study that FADS3 does exist under multiple protein isoforms depending on the mammalian tissues. These results will help further investigations to determine the physiological function of FADS3.
Assuntos
Ácidos Graxos Dessaturases/genética , Regulação Enzimológica da Expressão Gênica , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Linhagem Celular , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/análise , Ácidos Graxos Dessaturases/química , Ácidos Graxos Dessaturases/imunologia , Feminino , Humanos , Isoenzimas/análise , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/imunologia , Masculino , Camundongos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Especificidade da EspécieRESUMO
The hydroxysteroid dehydrogenase HSD1, identified in the proteome of oil bodies from mature Arabidopsis seeds, is encoded by At5g50600 and At5g50700, two gene copies anchored on a duplicated region of chromosome 5. Using a real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) approach, the accumulation of HSD1 mRNA was shown to be specifically and highly induced in oil-accumulating tissues of maturing seeds. HSD1 mRNA disappeared during germination. The activity of HSD1 promoter and the localization of HSD1 transcripts by in situ hybridization were consistent with this pattern. A complementary set of molecular and genetic analyses showed that HSD1 is a target of LEAFY COTYLEDON2, a transcriptional regulator able to bind the promoter of HSD1. Immunoblot analyses and immunolocalization experiments using anti-AtHSD1 antibodies established that the pattern of HSD1 deposition faithfully reflected mRNA accumulation. At the subcellular level, the study of HSD1:GFP fusion proteins showed the targeting of HSD1 to the surface of oil bodies. Transgenic lines overexpressing HSD1 were then obtained to test the importance of proper transcriptional regulation of HSD1 in seeds. Whereas no impact on oil accumulation could be detected, transgenic seeds exhibited lower cold and light requirements to break dormancy, germinate and mobilize storage lipids. Interestingly, overexpressors of HSD1 over-accumulated HSD1 protein in seeds but not in vegetative organs, suggesting that post-transcriptional regulations exist that prevent HSD1 accumulation in tissues deprived of oil bodies.
Assuntos
11-beta-Hidroxiesteroide Desidrogenases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Sementes/metabolismo , Triglicerídeos/biossíntese , 11-beta-Hidroxiesteroide Desidrogenases/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Sementes/genética , Sementes/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Seed oil bodies (OBs) are intracellular particles storing lipids as food or biofuel reserves in oleaginous plants. Since Brassica napus OBs could be easily contaminated with protein bodies and/or myrosin cells, they must be purified step by step using floatation technique in order to remove non-specifically trapped proteins. An exhaustive description of the protein composition of rapeseed OBs from two double-zero varieties was achieved by a combination of proteomic and genomic tools. Genomic analysis led to the identification of sequences coding for major seed oil body proteins, including 19 oleosins, 5 steroleosins and 9 caleosins. Most of these proteins were also identified through proteomic analysis and displayed a high level of sequence conservation with their Arabidopsis thaliana counterparts. Two rapeseed oleosin orthologs appeared acetylated on their N-terminal alanine residue and both caleosins and steroleosins displayed a low level of phosphorylation.
Assuntos
Brassica napus/química , Corpos de Inclusão/química , Proteínas de Armazenamento de Sementes/análise , Sementes/química , Sequência de Aminoácidos , Arabidopsis/genética , Brassica napus/genética , Brassica rapa/química , Brassica rapa/genética , Proteínas de Ligação ao Cálcio/análise , Immunoblotting , Dados de Sequência Molecular , Filogenia , Proteínas de Plantas/análise , Processamento de Proteína Pós-Traducional , Proteínas de Armazenamento de Sementes/química , Proteínas de Armazenamento de Sementes/genética , Alinhamento de SequênciaRESUMO
Proteomic approaches on lipid bodies have led to the identification of proteins associated with this compartment, showing that, rather than the inert fat depot, lipid droplets appear as complex dynamic organelles with roles in metabolism control and cell signaling. We focused our investigations on caleosin [Arabidopsis thaliana caleosin 1 (AtClo1)], a minor protein of the Arabidopsis thaliana seed lipid body. AtClo1 shares an original triblock structure, which confers to the protein the capacity to insert at the lipid body surface. In addition, AtClo1 possesses a calcium-binding domain. The study of plants deficient in caleosin revealed its involvement in storage lipid degradation during seed germination. Using Saccharomyces cerevisiae as a heterologous expression system, we investigated the potential role of AtClo1 in lipid body biogenesis and filling. The green fluorescent protein-tagged protein was correctly targeted to lipid bodies. We observed an increase in the number and size of lipid bodies. Moreover, transformed yeasts accumulated more fatty acids (+46.6%). We confirmed that this excess of fatty acids was due to overaccumulation of lipid body neutral lipids, triacylglycerols and steryl esters. We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote overaccumulation of storage lipids in yeast oil bodies.
Assuntos
Arabidopsis/genética , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação ao Cálcio/genética , Metabolismo dos Lipídeos , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fusão Gênica Artificial , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Organelas/metabolismo , Organelas/ultraestrutura , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
Oleosins are hydrophobic proteins from oleaginous seeds, surrounding and stabilizing oil bodies. They are known to display interesting interfacial properties. Specific sera were raised against four different A. thaliana oleosins and used in dot-blot assays for oleosin quantification. These assays were used to set up extraction of oleosins from A. thaliana seeds. One mixture of chloroform/methanol gave optimal oleosin extraction. Extracted proteins represented 9% of seed proteins and were identified by immunoblot and proteomic analyses. Oleosins accounted for 79% of the extracted proteins. This simple one-step procedure allows selective extraction and concentration of oleosins from seeds without tedious oil body purification. Oleosin extract was indeed used to demonstrate the presence of the rare oleosin S5 in mature seeds. Moreover, this method will be useful to investigate the potential use of oleosins as emulsifier and to question their possible allergenicity.
Assuntos
Proteínas de Arabidopsis/análise , Proteínas de Arabidopsis/química , Arabidopsis/química , Clorofórmio/química , Immunoblotting , Metanol/química , Óleos de Plantas/metabolismo , Sementes/química , SolubilidadeRESUMO
We have investigated the covalent and secondary solution structure of caleosin, a 27-kDa protein also called ATS1 or AtClo1 (At4g26740) found within Arabidopsis thaliana seed lipid bodies. The native protein was partly phosphorylated at S225. Purified bacterially expressed caleosin (recClo) was not phosphorylated; cysteine residues C221 and C230 were connected by a disulfide bridge. In solution it exists as a mixture of predominant monomers and covalent dimers. We have used recClo as a model for the study of AtClo1 secondary structure. recClo is folded in aqueous solution (16% alpha-helix, 29% beta-sheet), its secondary structure being dramatically influenced by the polarity of media, as deduced from CD spectra measured in the presence of increasing concentrations of various aliphatic alcohols.
Assuntos
Arabidopsis/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/ultraestrutura , Proteínas de Plantas/química , Proteínas de Plantas/ultraestrutura , Sementes/química , Sequência de Aminoácidos , Dicroísmo Circular , Espectrometria de Massas , Conformação Molecular , Dados de Sequência Molecular , Peso MolecularRESUMO
Yarrowia lipolytica contains five acyl-coenzyme A oxidases (Aox), encoded by the POX1 to POX5 genes, that catalyze the limiting step of peroxisomal beta-oxidation. In this study, we analyzed morphological changes of Y. lipolytica growing in an oleic acid medium and the effect of POX deletions on lipid accumulation. Protrusions involved in the uptake of lipid droplets (LDs) from the medium were seen in electron micrographs of the surfaces of wild-type cells grown on oleic acid. The number of protrusions and surface-bound LDs increased during growth, but the sizes of the LDs decreased. The sizes of intracellular lipid bodies (LBs) and their composition depended on the POX genotype. Only a few, small, intracellular LBs were observed in the mutant expressing only Aox4p (Deltapox2 Deltapox3 Deltapox5), but strains expressing either Aox3p or both Aox3p and Aox4p had the same number of LBs as did the wild type. In contrast, strains expressing either Aox2p or both Aox2p and Aox4p formed fewer, but larger, LBs than did the wild type. The size of the LBs increased proportionately with the amount of triacylglycerols in the LBs of the mutants. In summary, Aox2p expression regulates the size of cellular triacylglycerol pools and the size and number of LBs in which these fatty acids accumulate.
Assuntos
Acil-CoA Oxidase/metabolismo , Metabolismo dos Lipídeos , Yarrowia/metabolismo , Acil-CoA Oxidase/genética , Ácidos Graxos/análise , Ácido Oleico/farmacologia , Yarrowia/crescimento & desenvolvimento , Yarrowia/ultraestruturaRESUMO
Till now, only scattered data are available in the literature, which describes the protein content of plant oil bodies. Especially, the proteins closely associated with the model plant Arabidopsis thaliana oil bodies have never been previously purified and characterized. Oil bodies have been purified using flotation techniques, combined with incubations under high salt concentration, in the presence of detergents and urea in order to remove non-specifically trapped proteins. The identity and integrity of the oil bodies have been characterized. Oil bodies exhibited hydrodynamic diameters close to 2.6 microm, and a ratio fatty acid-protein content near 20. The proteins composing these organelles were extracted, separated by SDS-PAGE, digested by trypsin, and their peptides were subsequently analyzed by nano-chromatography-mass spectrometry (nano-LC-MS/MS). This led to the identification of a limited number of proteins: four different oleosins, ATS1, a protein homologous to calcium binding protein, a 11-beta-hydroxysteroid dehydrogenase-like protein, a probable aquaporin and a glycosylphosphatidylinositol-anchored protein with no known function. The two last proteins were till now never identified in plant oil bodies. Structural proteins (oleosins) represented up to 79% of oil body proteins and the 18.5 kDa oleosin was the most abundant among them.
Assuntos
Proteínas de Arabidopsis/análise , Arabidopsis/química , Lipídeos/química , Organelas/química , Sementes/química , Arabidopsis/ultraestrutura , Fracionamento Celular , Eletroforese em Gel de Poliacrilamida , Peso MolecularRESUMO
In order to study the effects of saturated fatty acids on delta6-desaturase activity, rat hepatocytes in primary culture were incubated with lauric (C12:0), myristic (C14:0) or palmitic (C16:0) acids. After optimization, the standard in vitro conditions for the measurement of delta6-desaturase activity were as follows: 60 micromol x L(-1) alpha-linolenic acid (C18:3n-3), reaction time of 20 min and protein content of 0.4 mg. Data showed that cell treatment with 0.5 mmol x L(-1) myristic acid during 43 h specifically increased delta6-desaturase activity. This improvement, reproducible for three substrates of delta6-desaturase, i.e. oleic acid (C18:1n-9), linoleic acid (C18:2n-6) and alpha-linoleic acid (C18:3n-3) was dose-dependent in the range 0.1-0.5 mmol x L(-1) myristic acid concentration.